JP2005516980A - Novel immune effector compounds - Google Patents

Abstract

The present invention provides a compound comprising 2-deoxy-2-amino-β-D-glucopyranose (glucosamine) glycosidically bonded to a cyclic aminoalkyl group (aglycone group). The invention further provides a method of inducing an immune response using a compound of the invention in the presence or absence of an antigen. In addition, methods of treating diseases using the compounds of the present invention with or without an antigen are also provided by the present invention.

Description

  The present invention relates generally to immune effector compounds, their use as pharmaceutical compositions, their production methods, their use as preventive and / or therapeutic vaccines. More specifically, the present invention relates to a compound containing 2-deoxy-2-amino-β-D-glucopyranose (glucosamine) glycosidically bonded to a cyclic aminoalkyl group (aglycone group), and the compound as a pharmaceutical adjuvant. It relates to a method used as a system.

  Humoral immunity and cell-mediated immunity are two major immune response mechanisms in mammals. Humoral immunity includes the production of antibodies against foreign antigens. Antibodies are produced by B lymphocytes. Cell-mediated immunity includes the activation of T lymphocytes. T lymphocytes act on infected cells with foreign antigens, or stimulate other cells to act on infected cells. In the mammalian immune system, both immune mechanisms are important in combating disease. Humoral immunity is a major mechanism in protecting against pathogenic bacteria. In the case of viral diseases, cytotoxic T lymphocytes (CTL) appear to be crucial for protective immunity. Thus, effective vaccines preferably stimulate both mechanisms of the immune system to protect against disease.

  Vaccines present foreign antigens from disease-causing pathogens to a host so that the host can elicit a protective immune response. Vaccine antigens are often in an inactivated or attenuated form of the disease-causing microorganism. Due to the presence of insignificant components and antigens in inactivated or attenuated vaccines, great efforts have been made to refine the vaccine components. One such effort is to develop well-known synthetic antigens using chemical and recombinant methods. However, purifying and simplifying microbial vaccines also reduced performance. Low molecular weight synthetic antigens do not contain potentially harmful contaminant components, but often are not sufficiently immunogenic per se. Based on such findings, researchers have added immune system stimulating factors known as adjuvants to vaccine compositions to enhance the activity of vaccine components.

  Immune adjuvants, when administered to an individual or tested in vitro, increase the immune response to the antigen in the patient's body, or increase certain activities of cells from the immune system. Compound. A large number of compounds with varying degrees of adjuvant activity have been prepared and tested (eg Shimizu et al., 1985; Bulusu et al., 1992; Ikeda et al., 1993; Shimizu et al., 1994; Shimizu et al., 1995) See Miyajima et al., 1996). However, these adjuvant systems, as well as other conventional adjuvant systems, are often toxic and / or unstable and / or have an unacceptably low immune promoting effect.

  At present, the only adjuvant approved for human use in the United States is alum (a group of aluminum salts, specifically aluminum hydroxide, aluminum phosphate, etc.). Vaccine antigens are formulated in this alum. Particulate carriers such as alum have been reported to promote macrophage uptake, processing and presentation of soluble antigens. However, alum is not without side effects and unfortunately is limited to humoral (antibody) immunity.

  The discovery and development of an effective adjuvant system is extremely important in improving the efficacy and safety of existing and future vaccines. Accordingly, there remains a need for new and improved adjuvant systems that enable the development of next generation synthetic vaccines, especially those that operate both mechanisms of the immune system. The present invention addresses these needs as well as other needs.

  The compounds of the present invention are immune effector molecules that promote the humoral and cell-mediated immune responses of vaccines against antigens. This compound can generally be said to belong to the class of cyclic AGP compounds. AGP represents aminoalkyl glucosaminidolinic acid. The term “cyclic AGP” refers to azacycloalkylglucosaminidolinic acid or (azacycloalkyl) alkylglucosaminidolinic acid in which 2-deoxy-2-amino-β-D-glucopyranose (glucosamine) is azacycloalkyl Means a glycoside bond to a group or an (azacycloalkyl) alkyl group (aglycone group).

で表わされる化合物と、その薬理学的に許容可能な塩である（ただし、Xは、-O-または-NH-であり；Yは、-O-または-S-であり；R¹、R²、R³は、それぞれ独立に、(C₉〜C₁₄)アシル基（飽和したアシル基、不飽和のアシル基、分枝状のアシル基が含まれる）であり；、R⁴は、-Hまたは-PO₃R⁷R⁸（ただしR⁷とR⁸は、それぞれ独立に、-Hまたは(C₁〜C₄)脂肪族基である）であり；R⁵は、-H、-CH₃、-PO₃R⁹R¹⁰（ただしR⁹とR¹⁰は、それぞれ独立に、-Hと(C₁〜C₄)脂肪族基の中から選択する）のいずれかであり；R⁶は、H、OH、(C₁〜C₄)オキシ脂肪族基、-PO₃R¹¹R¹²、-OPO₃R¹¹R¹²、-SO₃R¹¹、-OSO₃R¹¹、-NR¹¹R¹²、-SR¹¹、-CN、-NO₂、-CHO、-CO₂R¹¹、-CONR¹¹R¹²-（ただしR¹¹とR¹²は、それぞれ独立に、-Hと(C₁〜C₄)脂肪族基の中から選択する）のいずれかであり；ただしR⁴とR⁵の一方はリン含有基であって、R⁴が-PO₃R⁷R⁸である場合には、R⁵は-PO₃R⁹R¹⁰以外であり； “*1〜3”と“**”は、キラル中心を表わし；
n、m、p、qは、それぞれ独立に、0〜6の整数を表わすが、pとmの和は0〜6である）。 The compounds of the present invention contain 2-deoxy-2-amino-β-D-glucopyranose (glucosamine) glycosidically linked to a cyclic aminoalkyl group (aglycone group). In this compound, the 4th and 6th positions of the glucosamine ring are phosphorylated, and the 2nd and 3rd positions of the aglycone nitrogen and the glucosamine ring are acylated with alkanoyloxytetradecanoyl residues. The compounds of the present invention generally have the general formula (I):

And a pharmacologically acceptable salt thereof, wherein X is —O— or —NH—; Y is —O— or —S—; R ¹ , R is ^2, R ^3, each independently a (C ₉ -C ₁₄₎ acyl group (saturated acyl group, an acyl group of the unsaturated, includes branched acyl group);, R ⁴ is - H or —PO ₃ R ⁷ R ⁸ (wherein R ⁷ and R ⁸ are each independently —H or (C ₁ -C ₄ ) aliphatic group); R ⁵ is —H, —CH ₃ , -PO ₃ R ⁹ R ¹⁰ (wherein R ⁹ and R ¹⁰ are each independently selected from -H and (C ₁ -C ₄ ) aliphatic groups); R ⁶ is _{, H, OH, (C 1} ~C 4) oxy aliphatic _{^{group, -PO 3 R 11 R 12,}} -OPO 3 R 11 R 12, -SO 3 R 11, -OSO 3 R 11, -NR 11 R 12 _{^{, -SR 11, -CN, -NO 2}} , -CHO, -CO 2 R 11, -CONR 11 R 12 - ( provided that R ¹¹ and R ¹² are each independently, -H and (C ₁ ~C ₄₎ Select from among aliphatic groups) Ri proviso one of R ⁴ and R ⁵ is a phosphorus-containing group, when R ⁴ is -PO ₃ R ⁷ R ⁸ is, R ⁵ is other than _{^{-PO 3 R 9 R 10; "}} * “1-3” and “**” represent chiral centers;
n, m, p and q each independently represents an integer of 0 to 6, but the sum of p and m is 0 to 6).

、
N-[(R)-3-ドデカノイルオキシテトラデカノイル]-(S)-2-ピロリジニルメチル 2-デオキシ-4-O-ホスホノ-2-[(R)-3-ドデカノイルオキシテトラデカノイルアミノ]-3-O-[(R)-3-ドデカノイルオキシテトラデカノイル]-β-D-グルコピラノシド（一般式III）：

、
N-[(R)-3-デカノイルオキシテトラデカノイル]-(S)-2-ピロリジニルメチル 2-デオキシ-4-O-ホスホノ-2-[(R)-3-デカノイルオキシテトラデカノイルアミノ]-3-O-[(R)-3-デカノイルオキシテトラデカノイル]-β-D-グルコピラノシド（一般式IV）：

、
ならびにその薬理学的に許容可能な塩である。 In some embodiments of the compounds according to the invention, X and Y are each oxygen, R ⁴ is PO ₃ R ⁷ R ⁸ , R ⁵ and R ⁶ are H, n, m, p, q Is an integer from 0 to 3. In a further preferred embodiment, R ⁷ and R ⁸ are —H. In an even more preferred embodiment, n is 1, m is 2, and p and q are 0. In a more preferred embodiment, R ¹ , R ² , R ³ are (C ₉ -C ₁₃ ) acyl groups (most preferred are (C ₁₀ -C ₁₂ ) acyl groups). In a more preferred embodiment, * 1-3 are in the R configuration, Y is in the equator conformation, and ** is in the S configuration. Particularly preferred is N-[(R) -3-tetradecanoyloxytetradecanoyl]-(S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2-[(R)- 3-tetradecanoyloxytetradecanoylamino] -3-O-[(R) -3-tetradecanoyloxytetradecanoyl] -β-D-glucopyranoside (formula II):

,
N-[(R) -3-Dodecanoyloxytetradecanoyl]-(S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2-[(R) -3-dodecanoyloxytetra Decanoylamino] -3-O-[(R) -3-dodecanoyloxytetradecanoyl] -β-D-glucopyranoside (general formula III):

,
N-[(R) -3-decanoyloxytetradecanoyl]-(S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2-[(R) -3-decanoyloxytetra Decanoylamino] -3-O-[(R) -3-decanoyloxytetradecanoyl] -β-D-glucopyranoside (formula IV):

,
As well as pharmacologically acceptable salts thereof.

  The present invention also provides a pharmaceutical composition comprising a compound represented by the above general formula and special formula. This pharmaceutical composition can be combined with various antigens and made into various formulations known to those skilled in the art.

  The compounds of the present invention are also effective in methods of inducing an immune response in a patient. This method comprises the operation of administering to a patient a therapeutically effective amount of one or more compounds according to the invention, preferably in the form of a pharmaceutical composition also comprising a pharmacologically acceptable base. Yes.

  The present invention also includes a method for treating a mammal suffering from, or susceptible to, a pathogenic bacterial infection, cancer, or autoimmune disease. This method comprises the operation of administering to a mammal a therapeutically effective amount of one or more compounds according to the invention, preferably in the form of a pharmaceutical composition also comprising a pharmacologically acceptable base. It is out.

  Furthermore, the present invention also includes a method of treating a disease or condition that is ameliorated by the production of nitric oxide in a patient. This method contacts a patient with an effective amount of one or more compounds according to the present invention, or an effective amount of a composition comprising one or more compounds according to the present invention and a pharmaceutically acceptable base. Includes operations to let you. In some embodiments, the compounds of the invention can be administered 48 hours prior to ischemia, administered up to 48 hours after ischemia, or administered during ischemia.

Definition

The term “acyl” refers to a group from which the hydroxy portion of an organic acid has been removed. Acyl thus includes, for example, acetyl, propionyl, butyryl, decanoyl, pivaloyl. For example, “(C ₉ -C ₁₄ ) acyl” means an acyl group having 9 to 14 carbon atoms.

The term “aliphatic” used alone or as part of another substituent, unless otherwise indicated, has the specified number of carbon atoms (ie, C ₁ -C ₄ is 1-4 Means a straight or branched chain or cyclic hydrocarbon moiety (which means a carbon atom). Such hydrocarbon moieties also include moieties having both cyclic and chain elements. Note that the ring and chain may be completely saturated, or one or more sites may be unsaturated. Specific examples of the saturated hydrocarbon group include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, s-butyl, cyclopropyl, cyclopropylmethyl, methylene, ethylene, n-butylene and the like. Can be mentioned. An unsaturated alkyl group is an alkyl group having one or more double bonds and / or triple bonds. Specific examples of the unsaturated aliphatic group include vinyl, 2-propenyl, crotyl, -2- (butadienyl), 1-propynyl, 3-propynyl and the like.

  The term “oxyaliphatic” means a group in which an aliphatic group is attached to the remainder of the molecule through an oxygen atom.

  Each of the above terms (eg, “alkyl”, “acyl”) is intended to include both substituted and unsubstituted forms of the moiety.

A variety of substituents for the aliphatic group can be selected from a range of 0 to (2m ′ + 1) (where m ′ is the total number of carbon atoms contained in the substituent). Substituents to be selected are -OR ', = O, = S, = NR', = N-OR ', -NR'R ", -SR', -halogen, -SiR'R" R "', -OC (O) R', -C (O) R ', -CO ₂ R', -CONR'R ", -OC (O) NR'R", -NR "C (O) R ', -NR'-C (O) NR "R"', -NR "C (O) ₂ R', -NH-C (NH ₂ ) = NH, -NR'C (NH ₂ ) = NH, -NH- C (NH ₂ ) = NR ′, —S (O) R ′, —S (O) ₂ R ′, —S (O) ₂ NR′R ″, —CN, —NO _{2 and the} like. R ′, R ″ and R ″ ′ each independently represent hydrogen or an unsubstituted (C ₁ -C ₄ ) aliphatic group. One skilled in the art will appreciate from the above description of substituents that the term “alkyl” includes groups such as haloalkyl (eg, —CF ₃ and —CH ₂ CF ₃ ).

  The term “halo” or “halogen” used alone or as part of another substituent means a fluorine atom, a chlorine atom, a bromine atom, an iodine atom unless otherwise specified. In the compound having a plurality of halogen substituents, the halogens may be the same as or different from each other.

  The term “pharmacologically acceptable salt” was prepared using a relatively non-toxic acid or base selected depending on what the individual substituents found in the compounds described herein are. It is meant to include salts of active compounds. When the compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by adding the desired base as it is or by adding it in an appropriate inert solvent. . Specific examples of the pharmaceutically acceptable base addition salt include sodium salt, potassium salt, calcium salt, ammonium salt, organic amino salt, magnesium salt and the like. When the compound of the present invention contains a relatively basic functional group, an acid addition salt can be obtained by adding the desired acid as it is or by adding it in a suitable inert solvent. it can. Specific examples of pharmacologically acceptable acid addition salts include inorganic acids (for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Salts derived from monohydrogen sulfate, hydroiodic acid, phosphorous acid, and relatively non-toxic organic acids (acetic acid, propionic acid, isobutyric acid, oxalic acid, maleic acid, malonic acid, benzoic acid, succinic acid, And salts derived from suberic acid, fumaric acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Examples include salts of amino acids (for example, alginate) and salts of organic acids (for example, glucuronic acid and galacturonic acid) (for example, Berge, SM et al., “Pharmaceutical salts”, Journal of Pharmaceutical Science, 1977, 66th). Volume, see pages 1-19). Some special compounds according to the invention contain both basic and acid functional groups, so that the compounds can be converted into base addition salts and acid addition salts.

  The neutral forms of the compounds according to the invention can be generated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. Although the parent form of this compound differs from the various salt forms in some physical properties (eg, solubility in polar solvents), in other respects as far as the purpose of this invention is concerned, the salt Is equivalent to the parental form.

  The present invention also provides compounds in the form of prodrugs in addition to salts. Prodrugs of the compounds described in this specification are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Furthermore, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch with a suitable enzyme or chemical reagent.

  Some compounds according to the invention can exist in unsolvated and solvated forms, including hydrated forms. In general, the form of a solvate is equivalent to the form of a non-solvate and is intended to be included within the scope of the present invention. Some compounds according to the invention can exist in polycrystalline or amorphous form. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

  Some compounds according to the invention contain asymmetric carbon atoms (optical centers) or double bonds. The racemates, diastereomers, geometric isomers and individual isomers are all intended to be included within the scope of the present invention.

The compounds of the present invention may also contain isotopes in proportions different from nature with respect to one or more of the atoms that constitute such compounds. For example, the compound can be labeled with a radioactive isotope such as tritium ( ³ H), iodine-125 ( ¹²⁵ I), carbon-14 ( ¹⁴ C). Any variation in which a compound according to the invention is replaced by an isotope is intended to be within the scope of the invention, regardless of whether the isotope is radioactive.

Introduction To improve vaccine safety, manufacturers avoid recombinant, subunit vaccines that avoid whole cell inactivated vaccines. The preparation of such safer vaccines removes foreign bacterial or viral components, leaving the minimal structure or epitope that is considered necessary for protective immunity. Such vaccines have improved safety because they have removed extraneous bacterial or viral components that are often found to be toxic and pyrogenic. However, whole cell vaccines are very effective because the same toxic components provide non-specific immune stimulation. Without additional immunostimulation, the immunogenicity of recombinant or subunit vaccines containing minimal structures or epitopes is often very weak.

  MPL (registered trademark) immunostimulant (Corixa), which is a disaccharide molecule derived from LPS of Salmonella Minnesota R595, has immunostimulatory properties. MPL (registered trademark) immunostimulant (monophosphoryl lipid A) is a structural derivative of lipid A (ie LPS) and has an improved therapeutic index compared to lipid A (the US patent for the structure of monophosphoryl lipid A) No. 4,987,237; see US Pat. Nos. 4,436,727 and 4,436,728 for the preparation of monophosphoryl lipid A). Other effective immunostimulants include 3-de-O-acylated monophosphoryl lipid A (3D-MPL). This is described in US Pat. No. 4,912,094. This compound is safe to administer to humans up to a dose of at least 20 μg / kg. However, in some patients, doses up to 10 μg / kg can cause increased body temperature, influenza-like symptoms, increased heart rate, and a slight decrease in blood pressure. By evaluating cell cultures and animals, MPL® immunostimulants remain parental in that they retain the ability to induce pyrogenic and inflammatory cytokines (eg, TNF-α, IL-8). It can be seen that the immunostimulatory activity of LPS is retained somewhat (although this property cannot be seen without a higher dose than the parent LPS). Thus, the need for an effective vaccine adjuvant is well understood. Ideally, such an adjuvant would promote a protective immune response without undesirable toxicity and pyrogenicity.

  In order to obtain immunostimulants with low pyrogenicity, synthetic molecules similar in structure to MPL® immunostimulants have been prepared. Such a novel molecule, collectively referred to as aminoalkyl glucosaminidolinic acid (APG), consists of an acylated glucose moiety attached to an acylated aminoalkyl group (Johnson et al., 1999, Bioorg. Med Chem. Lett., Vol. 9, pp. 2273-2278; PCT application WO 98/50399 and references cited therein). Each molecule has six fatty acid tails. These six are considered to be the optimal numbers that maximize the activity of the adjuvant. Design substitutions of various moieties within the aminoalkyl structure for optimal stability and solubility and incorporate them into the AGP. AGP can therefore be roughly divided into several families based on the structure of the aminoalkyl group. Initial biological assessments revealed that aminoalkyl motifs can dramatically affect the pyrogenic properties of AGP (US patent application serial number filed May 7, 1998). No. 09 / 074,720 and U.S. Pat. Nos. 6,113,918 and 6,303,347). As part of the initial screening process for synthetic adjuvant compounds, rabbit fever data were revealed. It was found that some compounds did not cause a fever response when administered intravenously at a rate of 10 μg / kg. Generally speaking, this same compound failed to induce detectable levels of inflammatory cytokines TNF-α or IL-1β in an in vitro cytokine induction assay for human peripheral blood. This document reports studies on the adjuvant properties of a class of AGPs that induce minimal activity in both rabbit pyrogen testing and in vitro cytokine assays.

で表わされる化合物と、その薬理学的に許容可能な塩が提供される（ただし、Xは、-O-または-NH-であり；Yは、-O-または-S-であり；R¹、R²、R³は、それぞれ独立に、(C₉〜C₁₄)アシル基（飽和したアシル基、不飽和のアシル基、分枝状のアシル基が含まれる）であり；、R⁴は、-Hまたは-PO₃R⁷R⁸（ただしR⁷とR⁸は、それぞれ独立に、-Hまたは(C₁〜C₄)脂肪族基である）であり；R⁵は、-H、-CH₃、-PO₃R⁹R¹⁰（ただしR⁹とR¹⁰は、それぞれ独立に、-Hと(C₁〜C₄)脂肪族基の中から選択する）のいずれかであり；R⁶は、H、OH、(C₁〜C₄)オキシ脂肪族基、-PO₃R¹¹R¹²、-OPO₃R¹¹R¹²、-SO₃R¹¹、-OSO₃R¹¹、-NR¹¹R¹²、-SR¹¹、-CN、-NO₂、-CHO、-CO₂R¹¹、-CONR¹¹R¹²-（ただしR¹¹とR¹²は、それぞれ独立に、-Hと(C₁〜C₄)脂肪族基の中から選択する）のいずれかであり；ただしR⁴とR⁵の一方はリン含有基であって、R⁴が-PO₃R⁷R⁸である場合には、R⁵は-PO₃R⁹R¹⁰以外であり； “*1〜3”と“**”は、キラル中心を表わし；
n、m、p、qは、それぞれ独立に、0〜6の整数を表わすが、pとmの和は0〜6である）。 Compounds and Compositions According to the present invention, the general formula (I):

And a pharmacologically acceptable salt thereof, wherein X is —O— or —NH—; Y is —O— or —S—; R ¹ , R ² and R ³ are each independently a (C ₉ -C ₁₄ ) acyl group (including a saturated acyl group, an unsaturated acyl group, a branched acyl group); and R ⁴ is , -H or -PO ₃ R ⁷ R ^{8, where} R ⁷ and R ⁸ are each independently -H or (C ₁ -C ₄ ) aliphatic groups; R ⁵ is -H, -CH _3, -PO ₃ R ⁹ R ¹⁰ (provided that R ⁹ and R ¹⁰ are each independently, -H and (C ₁ -C ₄₎ selected from among aliphatic group) be either; R _{^6, H, OH, (C 1} ~C 4) oxy aliphatic _{^{group, -PO 3 R 11 R 12,}} -OPO 3 R 11 R 12, -SO 3 R 11, -OSO 3 R 11, -NR 11 ^{R 12, -SR 11, -CN,} -NO 2, -CHO, -CO 2 R 11, -CONR 11 R 12 - ( provided that R ¹¹ and R ¹² are each independently, -H and (C ₁ -C ₄₎ selected from among aliphatic group) noise Be either the proviso one of R ⁴ and R ⁵ is a phosphorus-containing group, when R ⁴ is -PO ₃ R ⁷ R ⁸ is, R ⁵ is other than -PO ₃ R ⁹ R ^10; “* 1-3” and “**” represent chiral centers;
n, m, p and q each independently represent an integer of 0 to 6, but the sum of p and m is 0 to 6).

  The hexopyranosides of general formula (I) are shown in the gluco configuration, but other glycosides are also within the scope of the present invention. For example, glycopyranoside is included within the scope of the present invention, including other hexopyranosides (eg, allo, altro, manno, glo, id, galacto, taro).

In the above general formula, the configuration of the 3 ′ stereogenic center (indicated as “* 1”, “* 2”, “* 3”) to which the normal fatty acyl residue is bonded is R configuration or S configuration. R configuration is preferred. The absolute stereochemical structure of the carbon atom of the cyclic aglycone unit (“**”) in which R ⁶ and the glucosamine unit are bonded directly or indirectly can be the R configuration or the S configuration. In the above general formula, Y can have an equator conformation or an axial conformation, but the equator conformation is preferred. All stereoisomers, enantiomers, diastereomers, and mixtures thereof are intended to be within the scope of the present invention.

を有するβ-D-グルコサミニド 4-リン酸2-ピロリジニルメチルである。 In a preferred embodiment of the invention, X and Y are —O; R ⁴ is phosphono; R ⁵ and R ⁶ are H; n, m, p, q are integers from 0 to 3; Preferably it is 0-2. Most preferably, the integer n is 1, the integer m is 2, and the integers p and q are 0. In this preferred embodiment, the compounds of the invention have the general formula (V):

Β-D-glucosaminide 4-phosphate 2-pyrrolidinylmethyl having the formula:

  In one preferred embodiment of the invention, the configuration of the 3 ′ stereogenic center to which the normal fatty acyl is attached (“* 1-3”) is the R configuration, Y is the equatorial conformation, and the stereogenic center of pyrrolidine. The absolute stereochemical structure of (“**”) is the S configuration.

で表わされるN-[(R)-3-テトラデカノイルオキシテトラデカノイル]-(S)-2-ピロリジニルメチル 2-デオキシ-4-O-ホスホノ-2-[(R)-3-テトラデカノイルオキシテトラデカノイルアミノ]-3-O-[(R)-3-テトラデカノイルオキシテトラデカノイル]-β-D-グルコピラノシドとその薬理学的に許容可能な塩、一般式（III）：

で表わされるN-[(R)-3-ドデカノイルオキシテトラデカノイル]-(S)-2-ピロリジニルメチル 2-デオキシ-4-O-ホスホノ-2-[(R)-3-ドデカノイルオキシテトラデカノイルアミノ]-3-O-[(R)-3-ドデカノイルオキシテトラデカノイル]-β-D-グルコピラノシドとその薬理学的に許容可能な塩、一般式（IV）：

で表わされるN-[(R)-3-デカノイルオキシテトラデカノイル]-(S)-2-ピロリジニルメチル 2-デオキシ-4-O-ホスホノ-2-[(R)-3-デカノイルオキシテトラデカノイルアミノ]-3-O-[(R)-3-デカノイルオキシテトラデカノイル]-β-D-グルコピラノシドとその薬理学的に許容可能な塩である。 Particularly preferred embodiments are those of the general formula (II):

N-[(R) -3-tetradecanoyloxytetradecanoyl]-(S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2-[(R) -3- Tetradecanoyloxytetradecanoylamino] -3-O-[(R) -3-tetradecanoyloxytetradecanoyl] -β-D-glucopyranoside and pharmacologically acceptable salts thereof, represented by the general formula (III ):

N-[(R) -3-dodecanoyloxytetradecanoyl]-(S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2-[(R) -3-dodeca Noyloxytetradecanoylamino] -3-O-[(R) -3-dodecanoyloxytetradecanoyl] -β-D-glucopyranoside and pharmacologically acceptable salts thereof, general formula (IV):

N-[(R) -3-decanoyloxytetradecanoyl]-(S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2-[(R) -3-deca Noyloxytetradecanoylamino] -3-O-[(R) -3-decanoyloxytetradecanoyl] -β-D-glucopyranoside and pharmacologically acceptable salts thereof.

Compound Preparation The compounds of the present invention are described in Johnson et al., 1999, Bioorg. Med. Chem. Lett., Vol. 9, pp. 2273-2278, and PCT application WO 98/50399 and references cited therein. Can be prepared using the method outlined in. In general, the synthetic methods described in the above references can be widely applied to the preparation of compounds having various acyl groups and substituents. One skilled in the art can modify the method described therein to use another acylating agent, or use a commercially available material with an appropriate acyl group attached. It will be understood that you can start.

Evaluation of Compounds The compounds described herein can be evaluated in a variety of assays to select compounds with appropriate drug transport profiles. For example, US Pat. No. 6,013,640 describes a model animal suitable for evaluating the cardioprotective effects of the compounds described herein. In the examples below, an assay for assessing the pyrogenicity of the compounds of the present invention and an assay for assessing the pro-inflammatory effects of the compounds are also presented.

  The present invention further provides pharmaceutical compositions in which one or more pharmacologically acceptable bases are mixed with the compounds provided herein. The appropriate base depends on the disease to be treated and the route of administration. Therefore, the description regarding the base will be described later in relation to the utilization method.

Pharmaceutical compositions and methods of use thereof According to one embodiment of the present invention, there is provided a pharmaceutical composition comprising a compound of the present invention and a pharmacologically acceptable base. The compound is present in a therapeutically effective amount. It is the amount of compound necessary to achieve the desired effect with respect to the treatment of the disease or condition, ie the amount necessary to realize the biological event. This pharmaceutical composition functions as an adjuvant when administered simultaneously with the antigen.

  There are two types of compositions of the present invention. One is a composition formulated alone or together with a vaccine or other active agent to administer the active compound directly to the patient without dilution, and the other is a large amount of diluent (eg, water, It is a more concentrated composition formulated so that it can be diluted later so that it does not have to be transported and / or stored. In general, a pharmaceutical composition of the invention for administration directly (ie, undiluted) to a patient will contain a therapeutically effective amount of one or more of the above compounds. This amount will vary depending on both what the individual compounds for treatment are and what therapeutic effect is desired. A more concentrated composition will contain the compound according to the invention in an appropriate amount in such a composition.

  The pharmacologically acceptable base for preparing the pharmaceutical composition can be solid or liquid. Solid preparations include powders, tablets, pills, capsules, cachets, suppositories, dispersible particles, and the like. The solid base can be one or more substances that also function as diluents, flavoring agents, binders, preservatives, tablet disintegrants, and capsule materials.

  In the case of powders, the base is a finely divided solid which is mixed with the finely divided active ingredient. In the case of tablets, the active ingredient is mixed in a suitable proportion with a base having the necessary binding properties and compressed to the desired shape and size.

  Solid form compositions can also be prepared by spray drying an aqueous formulation of the active adjuvant (eg, in the form of a salt) or by lyophilizing and then grinding with excipients.

  Suitable bases for the solid composition according to the invention include, for example, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth gum, methylcellulose, sodium carboxymethylcellulose, low melting wax, Examples include cocoa butter. The term “preparation” includes formulations consisting of the active compound and a capsule material as a base for capsule formation. Since the active ingredient is surrounded by the base in the capsule (may or may not contain other bases), the base is integrated with the active ingredient. Similarly, cachets and lozenges are included in the term preparation. Tablets, powders, capsules, pills, cachets, lozenges can be used in solid dosage forms suitable for oral administration.

  To prepare a suppository, a low melting wax (eg, fatty acid glyceride or cocoa butter) is first melted and the active component is dispersed homogeneously therein, with stirring. The melted homogeneous mixture is then poured into a suitably sized mold and allowed to solidify by cooling.

  Liquid form preparations include solutions, suspensions, emulsions and the like, and specifically include aqueous solutions or water / propylene glycol solutions. For parenteral injection, the liquid preparation can be made into a solution in aqueous polyethylene glycol solution. In some embodiments, the pharmaceutical composition is a stable emulsion formulation (eg, a water-in-oil emulsion or an oil-in-water emulsion) or an aqueous formulation. Such a preparation preferably contains one or more surfactants. In such emulsions, suitable surfactants well known to those skilled in the art can be used. In one embodiment, the composition is in the form of a micelle dispersion comprising at least one surfactant. Surfactants effective in such micelle dispersions include phospholipids. Specific examples of phospholipids include diacyl phosphatidyl glycerol (eg, dimyristoyl phosphatidyl glycerol (DPMG), dipalmitoyl phosphatidyl glycerol (DPPG), distearoyl phosphatidyl glycerol (DSPG)); diacyl phosphatidyl cholines (eg, dimyristoyl phosphatidyl choline (DPMC)) , Dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC); diacyl phosphatidic acid (eg, dimyristoyl phosphatidic acid (DPMA), dipalmitoyl phosphatidic acid (DPPA), distearoyl phosphatidic acid (DSPA); For example, dimyristoyl phosphatidylethanolamine (DPME), dipalmitoyl phosphatidyl eta Nolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), etc. Other specific examples include derivatives of ethanolamine (eg phosphatidylethanolamine or cephalin as described above), serine (eg phosphatidylserine), 3′- Examples thereof include, but are not limited to, O-lysylglycerol (eg, 3′-O-lysylphosphatidylglycerol).

  Aqueous solutions suitable for oral administration can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizers, thickening agents as desired. Aqueous suspensions suitable for oral administration disperse the finely divided active ingredient in water with viscous materials (eg, natural or synthetic rubbers, resins, methylcellulose, sodium carboxymethylcellulose) and other well known suspending agents. Can be prepared.

  Some solid form preparations convert to liquid preparation forms for oral administration shortly before use. Such liquid forms include solutions, suspensions, emulsions and the like. Such preparations can contain, in addition to the active ingredient, colorants, flavors, stabilizers, buffers, artificial sweeteners, natural sweeteners, dispersants, thickeners, solubilizers, and the like.

  The pharmaceutical preparation is preferably in unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. Unit dosage forms can be packaged preparations. This package contains divided quantities of the preparation such as packed tablets, capsules, bottles or powders in ampoules. As the unit dosage form, capsules, tablets, cachets, and lozenges can be used as they are. Alternatively, any of these can be packaged in an appropriate number.

  Thus, the adjuvant system of the present invention is used to make and use vaccines and other immune enhancing compositions to treat or prevent diseases in mammals, preferably humans, for example by inducing active immunity against antigens. Especially suitable. Vaccine preparation is a well-established technique, and general information on vaccine preparation and formulation is readily available from a variety of literature. One example is the New Trends and Developmencs in Vaccines exhibition edited by Voller et al., University Park Press, Baltimore, Maryland, USA, 1978.

  In one embodiment, the antigen contained in the vaccine composition of the invention is a peptide, polypeptide, or an immunogenic portion thereof. The “immunogenic portion” in this specification is the portion of the protein that is recognized (ie, specifically binds) by the B cell and / or T cell surface antigen receptors. Such immunogenic portions generally comprise at least 5 amino acid residues of the antigen protein or variant thereof. The number of amino acid residues is more preferably at least 10 and even more preferably at least 20.

The immunogenic portion of an antigen polypeptide can generally be identified using well-known methods. Such methods are summarized in, for example, Paul, “Fundamental Immunology”, 3rd edition, pages 243-247 (Raven Press, 1993) and references cited therein. Such methods include screening for whether the polypeptide has the ability to react with antigen-specific antibodies, antisera, T cell lines, T cell clones. As used herein, antisera and antibodies specifically bind to an antigen (ie, react with that protein in an ELISA or other immunoassay but do not cause a detectable reaction with an irrelevant protein). Expressed “antigen-specific”. Such antisera and antibodies can be prepared as described herein utilizing well known methods. The immunogenic portion of the protein reacts with such antisera and / or T cells (eg, in an ELISA and / or T cell response assay) at a level not substantially lower than the reactivity of the full length polypeptide. It is a part to do. This immunogenic portion can react in such assays at a level similar to or greater than the reactivity of the full-length polypeptide. Such screening can generally be performed by methods well known to those skilled in the art. Such methods are described, for example, in Harlow and Lane, “Antibodies: A Labratory Manual”, Cold Spring Harbor Laboratory, 1988. For example, the polypeptide is immobilized on a solid support, contacted with the patient's serum, and the antibodies in the serum are bound to the immobilized polypeptide. The unbound serum is then removed and the bound antibody is detected using, for example, protein A labeled with ¹²⁵ I.

  Peptide antibodies and polypeptide antibodies are prepared using any of a variety of well-known methods. A recombinant polypeptide encoded by a DNA sequence can be readily prepared from the isolated DNA sequence using any of a variety of expression vectors known to those of skill in the art. Expression can be achieved in any suitable host cell transformed with an expression vector containing a DNA molecule encoding a recombinant polypeptide, or any suitable host cell transfected with such an expression vector. . Suitable host cells include prokaryotic cells, yeast, evolved eukaryotic cells (eg, mammalian cells and plant cells) and the like. The host cell used is preferably an E. coli, yeast, or mammalian cell system (eg COS or CHO).

  Portions and variants of protein antigens having less than about 100 amino acids (more typically less than about 50) can also be produced by synthetic means well known to those skilled in the art. For example, such polypeptides are synthesized using any of the available solid phase methods (eg, the Merrifield solid phase synthesis method in which amino acids are sequentially added to a growing amino acid chain). be able to. See Merrifield, J Am. Chem. Soc., 85, 2146-2149, 1963. Polypeptide automated synthesizers are available from manufacturers such as Perkin Elmer / Applied Biosystems Division (Foster City, Calif.) And can be operated according to the manufacturer's instructions.

  In some special embodiments, the polypeptide antigen used in the vaccine composition of the invention can be a fusion protein comprising two or more different polypeptides. Fusion partners, for example, help provide T helper epitopes (immune fusion partners), preferably T helper epitopes recognized by humans, or help proteins to be expressed at higher levels than raw recombinant proteins (Expression enhancer). Some preferred fusion partners are fusion partners that are both immune and promote expression. Other fusion partners can be selected to increase the solubility of the protein, or can be selected to target the intracellular compartment the protein desires. Yet another fusion partner includes an affinity tag that facilitates protein purification.

  Fusion proteins can generally be prepared using standard methods (eg, chemical coupling methods). The fusion protein is preferably expressed as a recombinant protein and is produced at a higher level in the expression system than the non-fusion protein. In short, DNA sequences encoding polypeptide elements can be assembled and ligated separately to form an appropriate expression vector. Ligating the 3 'end of a DNA sequence encoding one polypeptide element with or without a peptide linker to the 5' end of a DNA sequence encoding a second polypeptide element • Ensure that the frames are aligned with each other. This allows translation into a single fusion protein that retains the biological activity of both polypeptide elements.

  Peptide linker sequences can be utilized to separate the first and second polypeptide elements a sufficient distance so that each polypeptide can be folded into a secondary and tertiary structure. Such peptide linker sequences are incorporated into the fusion protein using methods well known in the art. Appropriate peptide linker sequences may be selected in view of the following: (1) flexible extension arrangements; (2) functional epitopes on the first or second polypeptide Inability to adopt secondary structures that may interact; (3) No hydrophobic or charged residues that may interact with the polypeptide functional epitope. Preferred peptide linker sequences contain glycine, asparagine and serine residues. Other nearly neutral amino acids (eg, threonine or alanine) can also be used in the linker sequence. Amino acid sequences that may be used as linkers include: Maratea et al., Gene, 40, 39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA, 83, 8258-8262. Page, 1986; those described in US Pat. Nos. 4,935,233 and 4,751,180. The linker sequence is generally from 1 to about 50 amino acids in length. If the first and second polypeptides have an unimportant N-terminal amino acid region that can be used to separate functional domains and prevent steric hindrance, a linker sequence is not necessary.

  In a preferred embodiment, the immunofusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926). The protein D derivative preferably comprises about the first third of the protein (eg 100-110 amino acids from the N-terminus). Protein D derivatives can be lipidated. In some preferred embodiments, the first 109 residues of the lipoprotein D fusion partner are included at the N-terminus, thus providing the polypeptide with additional exogenous T cell epitopes to increase the level of expression in E. coli. Increase (and thus function as an expression enhancer). The lipid tail presents the antigen in an optimal state to antigen-presenting cells. Other fusion partners include nonstructural proteins from influenza virus NS1 (hemagglutinin). Generally, the N-terminal 81 amino acids are used, but other fragments containing T helper epitopes can be used.

  In another embodiment, the immunofusion partner is a protein known as LYTA, or a portion thereof (preferably the C-terminal portion). LYTA is derived from S. pneumoniae. S. pneumoniae synthesizes N-acetyl-L-alanine amidase (encoded by the LytA gene) known as amidase LYTA (Gene, 43, 265-292, 1986). LYTA is an autolytic enzyme that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is important for its affinity for choline or certain choline analogs (eg DEAE). This property was developed to create an E. coli C-LYTA expression plasmid useful for fusion protein expression. Purification of hybrid proteins containing a C-LYTA fragment at the amino terminus has already been reported (see Biotechnology, Vol. 10, pages 795-798, 1992). In one preferred embodiment, a repeat portion of LYTA can be incorporated into the fusion protein. One repeat is found in the part starting at residue 178 in the C-terminal region. Particularly preferred repeat moieties include residues 188-305.

  In another embodiment of the invention, the adjuvant system described herein is used to prepare a DNA-based vaccine composition. A typical vaccine of this type contains DNA encoding one or more polypeptide antigens so that the antigen is generated in situ. The DNA may be present in any of a variety of delivery systems such as nucleic acid expression systems, bacterial expression systems, viral expression systems known to those skilled in the art. A number of gene delivery methods are known in the art. For example, there are methods described in Rolland, Crit. Rev. Therap. Drug Carrier Systems, Vol. 15, pp. 143-198, 1998, and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient's body (eg, appropriate promoters and termination signals). Bacterial delivery systems also include the administration of bacteria (eg, Calmette-Guerin) that express the immunogenic portion of the polypeptide on the cell surface or secrete such epitopes. In a preferred embodiment, the DNA is introduced using a viral expression system (eg, a poxvirus such as vaccinia virus, a retrovirus, an adenovirus). Viral expression systems generally use non-pathogenic (defective) replicable viruses. Specific examples of expression systems are disclosed, for example, Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA, 86, 317-321, 1989; Flexner et al., Ann. NY Acad. Sci. , 569, 86-103, 1989; Flexner et al., Vaccine, 8, 17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, 5,017,487; WO 89/01973; United States Patent 4,777,127; British Patent 2,200,651; European Patent 0,345,242; WO 91/02805; Berkner, Biotechniques, 6, 616-627, 1988; Rosenfeld et al., Science, 252, 431-434 Kolls et al., Proc. Natl. Acad. Sci. USA, 91, 215-219, 1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA, 90, 11498- 11502, 1993; Guzman et al., Circulation, 88, 2838-2848, 1993; Guzman et al., Cir. Res., 73, 1202-1207, 1993. Methods for incorporating DNA into such expression systems are well known to those skilled in the art.

  Alternatively, the DNA may be “naked”. This is described, for example, in Ulmer et al., Science, Vol. 259, pages 1745-1749, 1993, and outlined in Cohen, Science, Vol. 259, pages 1691-1692, 1993. To increase the uptake of naked DNA, the surface of biodegradable beads that are effectively carried into the cell can be coated with the DNA. It will be apparent that a vaccine may contain both a polynucleotide component and a polypeptide component if desired.

  It will also be apparent that the vaccine may contain pharmacologically acceptable salts of the desired polynucleotide and / or polypeptide antigens and / or carbohydrate antigens. For example, such salts can be prepared from pharmacologically acceptable non-toxic bases. Non-toxic bases include organic salts (eg primary amine salts, secondary amine salts, tertiary amine salts, basic amino acids) or inorganic salts (eg sodium salts, potassium salts, lithium salts, ammonium salts). Salt, calcium salt, magnesium salt).

  The adjuvant system of the present invention exhibits a strong adjuvant effect when administered in a wide range of doses as well as a wide range of ratios.

  The amount of antigen contained in each vaccine dose is generally such that it induces a protective immune response without the significant side effects of typical vaccines appearing. Such an amount will vary depending on the specific immunogen used and how it is presented. In general, each dose will contain about 1-1000 μg of protein, more typically about 2-100 μg, preferably 5-50 μg. Of course, dosage may vary depending on age, weight, type of treatment currently being performed (if any) and the nature of the antigen being administered.

  The immune activity of a given amount of vaccine composition according to the present invention can be readily determined, for example, by monitoring the titer of antibodies against the antigen used in the vaccine composition (Dalsgaard, K., Acta Veterinia Scandinavica, 69, pages 1-40, 1978). Another common method involves injecting various amounts of the vaccine composition into the skin of CD-1 mice and later collecting the serum from the mice and examining for anti-immunogenic antibodies (eg by ELISA). Contains. These and other similar methods will be apparent to those skilled in the art.

  Depending on whether the antigen is an infectious disease, an autoimmune disease, what symptoms it is, cancer, what is the pathogen, and what vaccine composition it is to treat Can be removed and / or isolated from almost any desired source. Antigens can be extracted from various viruses. Examples of viruses include influenza virus, feline leukemia virus, feline immunodeficiency virus, human HIV-1, human HIV-2, herpes simplex virus type 2, human cytomegalovirus, hepatitis A virus, hepatitis B virus, C Examples include hepatitis B virus, hepatitis E virus, RS virus, human papilloma virus, rabies virus, measles virus, and foot-and-mouth disease virus. Representative antigens can also be extracted from various bacteria. Examples of bacteria include anthrax, diphtheria, Lyme disease, malaria, tuberculosis, Leishmania, Trypanosoma cruzi, Ehrlichia and Candida, as well as protozoa (eg Babesia bovis and Plasmodium). and so on. Antigens are generally composed of natural or synthetic amino acids in the form of peptides, polypeptides, proteins, for example, but can also be composed of polysaccharides or mixtures of amino acids and polysaccharides. Representative antigens can be isolated from natural sources, synthesized by solid phase synthesis, or obtained by recombinant DNA methods.

  In another embodiment, tumor antigens are utilized in the vaccine composition of the present invention to prevent and / or treat cancer. Cancer cells often have antigens that are clearly distinguishable on their surface. Examples of antigens include cleaved epidermal growth factor, folate binding protein, epithelial mucin, melanoferrin, carcinoembryonic antigen, prostate specific membrane antigen, HER2-neu, which are for use in vaccines for cancer treatment Is a candidate. Because tumor antigens are or are associated with normal elements of the body, the immune system often fails to destroy tumor cells by producing an effective immune response against these antigens. is there. To achieve such a response, the adjuvant system described in this specification can be used. As a result, foreign proteins can enter the processing pathway of endogenous antigen, and T cells (CTL) that are cytolytic or cytotoxic are produced. This adjuvant effect promotes the production of antigen-specific CTLs that search for and destroy tumor cells that have tumor antigens on the surface used for immunization. Typical cancer types that can use this method are prostate cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, brain tumor, head and neck cancer, melanoma, leukemia, lymphoma, etc. .

  In another embodiment of the present invention, the adjuvant system of the present invention alone (ie without simultaneous administration of antigens) is used to strengthen the immune system, particularly for the treatment of chronic infectious diseases in patients with weak immunity. ) Can be administered. Representative examples of infectious diseases that may be used in the treatment or prevention of this method can be found in US Pat. No. 5,508,310. Strengthening the immune system in this way is also effective as a preventive measure to reduce the risk of nosocomial infection and / or post-operative infection.

  In another embodiment, for example, the vaccine composition is for an autoimmune disease because the antigen present in the vaccine composition is a foreign antigen rather than a foreign antigen. Examples of autoimmune diseases include type 1 diabetes, conventional organ-specific autoimmune diseases, neurological diseases, rheumatic diseases, psoriasis, connective tissue diseases, autoimmune cytopenias, and other autoimmune diseases. . Conventional organ-specific autoimmune diseases include thyroiditis (Graves + Hashimoto's thyroiditis), gastritis, adrenalitis (Addison adrenalitis), ovitis, primary biliary cirrhosis, myasthenia gravis, gonad failure, accessory gland failure Examples include hypothyroidism, alopecia, malabsorption syndrome, pernicious anemia, hepatitis, anti-receptor antibody disease, and vitiligo. Examples of neurological diseases include schizophrenia, Alzheimer's disease, depression, hypopituitarism, diabetes insipidus, dry syndrome, and multiple sclerosis. Rheumatoid / connective tissue diseases include rheumatoid arthritis, systemic lupus erythematosus (SLE) or lupus, scleroderma, polymyositis, inflammatory bowel disease, dermatomyositis, ulcerative colitis, Crohn's disease, vasculitis, psoriasis Examples include osteoarthritis, exfoliative psoriatic dermatitis, pemphigus vulgaris, and Sjogren's syndrome. Other autoimmune related diseases include autoimmune uveoretinitis, glomerulonephritis, post-myocardial infarction syndrome, pulmonary hemosiderinosis, amyloidosis, sarcoidosis, aphthous stomatitis, and others presented in this specification. Other immune-related diseases known in the relevant field.

  Any suitable base known to those skilled in the art can be used in the vaccine compositions of the invention, but the type of base will generally vary depending on what is desired the method of administration. The vaccine composition of the present invention can be administered by any suitable method (eg, local administration, oral administration, intranasal administration, intravenous administration, intracranial administration, intraperitoneal administration, intradermal administration, subcutaneous administration, intramuscular administration). Can also be formulated. For parenteral administration (eg, subcutaneous injection), the base often contains any of water, saline, alcohol, fat, wax, and buffer. For oral administration, the above bases are often used. Alternatively, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate can also be used as a solid base. Biodegradable microspheres (eg polyglycolate polylactate) can also be used as a base for the composition according to the invention. Suitable biodegradable microspheres are disclosed, for example, in US Pat. Nos. 4,897,268, 5,075,109, 5,928,647, 5,811,128, 5,820,883, 5,853,763, 5,814,344, and 5,942,252. The contents of these patent documents are incorporated in this specification in their entirety for reference. Modified hepatitis B core protein base systems are also suitable as bases. This is described in WO 99/40934 and the references cited therein, the entirety of which is incorporated herein by reference. Particulate protein complexes that can induce a host to induce a class I restricted cytotoxic T lymphocyte response (eg, those described in US Pat. No. 5,928,647, the contents of this patent document being incorporated by reference) It is also possible to use a base comprising the entirety of which is incorporated herein.

  In one exemplary embodiment, the vaccine formulation is administered to the mucosa to elicit an immune response, but is preferably administered to the palate, particularly the sublingual site. Administration to the palate will often be preferred over traditional parenteral delivery because it is simple because it is a non-invasive method of administration. Furthermore, this method provides a means of eliciting mucosal immunity. This is often difficult to achieve with traditional parenteral delivery. Also, mucosal immunity can protect against airborne pathogens and / or allergens. Another advantage of palatal administration is the provision of vaccines sublingually to ensure patient compliance, especially in pediatrics and areas where multiple injections are generally required over time, such as allergic sensitization therapy. There is a possibility of improvement.

  Further included in the vaccine composition are buffers (eg, neutral buffered saline, phosphate buffered saline, phosphate buffered w / o saline), carbohydrates (eg, glucose, mannose, sucrose, dextran). ), Mannitol, protein, polypeptide, amino acid (eg glycine), antioxidant, bacteriostatic agent, chelating agent (EDTA or glutathione), adjuvant (eg aluminum hydroxide), formulation isotonic with patient blood, hypotonic Solutes, suspending agents, thickeners, preservatives to make them slightly hypertonic. The vaccine composition of the present invention can also be encapsulated in liposomes using well known methods.

  Thus, in one embodiment, the vaccine composition is an aqueous formulation comprising an effective amount of one or more surfactants. For example, the composition can be in the form of a micelle dispersion containing at least one suitable surfactant (eg, a phospholipid surfactant). Specific examples of phospholipids include diacyl phosphatidyl glycerol (eg, dimyristoyl phosphatidyl glycerol (DPMG), dipalmitoyl phosphatidyl glycerol (DPPG), distearoyl phosphatidyl glycerol (DSPG)); diacyl phosphatidyl cholines (eg, dimyristoyl phosphatidyl choline (DPMC)) , Dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC); diacyl phosphatidic acid (eg, dimyristoyl phosphatidic acid (DPMA), dipalmitoyl phosphatidic acid (DPPA), distearoyl phosphatidic acid (DSPA); For example, dimyristoyl phosphatidylethanolamine (DPME), dipalmitoyl phosphatidyl eta Examples include nolamine (DPPE) and distearoylphosphatidylethanolamine (DSPE).

  Generally, the molar ratio of surfactant: adjuvant in the aqueous formulation is from about 10: 1 to about 1:10, but more commonly is from about 5: 1 to about 1: 5. However, any effective amount of surfactant can be used in the aqueous formulation to be optimal for a particular purpose.

  In another embodiment, the composition is an emulsion (eg, a water-in-oil emulsion or an oil-in-water emulsion). Such emulsions are generally well known to those skilled in the art.

The adjuvant system of the present invention can be used as the sole adjuvant system or can be administered in combination with other adjuvants or immune effectors. For example, adjuvants include oil-based adjuvants (eg, Freund's complete or incomplete adjuvant), liposomes, inorganic salts (eg, AlK (SO ₄ ) ₂ , AlNa (SO ₄ ) ₂ , AlNH ₄ (SO ₄ ), Silica, alum, Al (OH) ₃ , Ca ₃ (PO ₄ ) ₂ , kaolin, carbon), polynucleotide (eg, poly IC acid, poly AU acid), polymer (eg, nonionic block polymer, polyphosphazene, Cyanoacrylate, polymerase- (DL-lactide-co-glycoside) Adjuvants can also include certain natural substances such as lipid A and its derivatives, wax D from Mycobacterium tuberculosis, and corynebacterium Substances found in Parbum, Bordetella pertussis, Brucella), bovine serum albumin, diphtheria toxoid, tetanus toxoid Edestine, mussel hemocyanin, pseudomonas toxin A, choleragenoids, cholera toxin, pertussis toxin, viral proteins, eukaryotic proteins (eg, interferon, interleukin, tumor necrosis factor), etc. Can be obtained from natural sources or from recombinant sources according to methods well known to those skilled in the art Adjuvants obtained from recombinant sources include at least a fragment containing an immunostimulatory portion of the protein. Other known immunostimulatory macromolecules that can be used in the practice of the present invention include polysaccharides, tRNAs, non-metabolizable synthetic polymers (eg, polyvinylamine, polymethacrylic acid, polyvinylpyrrolidone, 4 ′, 4-diamino). Diphenylmethane-3,3'-dicarboxylic acid and 4-nitro-2-aminobenzoic acid (with relatively high molecular weight) polycondensation mixture (see Sela, M., Science, 166, pp. 1365–1374, 1969)), glycolipids, lipids And carbohydrates.

  In one embodiment, the adjuvant system is preferably designed to induce predominantly a Th1-type immune response. High levels of Th1-type cytokines (eg IFN-γ, TNF-α, IL-2, IL-12) tend to induce cell-mediated immune responses against the administered antigen. Conversely, high levels of Th2-type cytokines (eg, IL-4, IL-5, IL-6, IL-10) tend to induce humoral immunity. Applying the vaccines described in this specification will help patients support immune responses including Th1 and Th2 type responses. In a preferred embodiment in which a Th1 type response predominates, the level of Th1 type cytokines will be greater than the level of Th2 type cytokines. The levels of these cytokines can be easily assessed using standard assays. For a review of the cytokine family, see Mosmann and Coffman, Ann. Rev. Immunol., 7, pp. 145-173, 1989.

  For example, additional adjuvants used to elicit dominant Th1-type responses include, for example, a combination of monophosphoryl lipid A (eg, 3-de-O-acrylated monophosphoryl lipid A (3D-MPL) and an aluminum salt. MPL adjuvants are available from Corixa (Seattle, WA; see US Pat. Nos. 4,436,727, 4,877,611, 4,866,034, 4,912,094) (CpG dinucleotides are not methylated) CpG-containing oligonucleotides also induce predominantly Th1-type responses, such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488, US Patent Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, in Sato et al., Science, 273, 352, 1996. Included in vaccine compositions Other representative adjuvants that can be used include ISA 720 (Sepic, France), SAF (Chiron, CA, USA), ISCOMS (CSL), MF-59 (Chiron), Detox (registered trademark) ) Adjuvant (Corixa, Hamilton, Mass.).

  The compositions described herein can be administered as part of a sustained release formulation (ie, a capsule, sponge, gel, etc. formulation that slowly releases the compound after administration (eg, composed of polysaccharides)). Such formulations can generally be prepared using well-known methods (see eg Coombes et al., Vaccine, Vol. 14, pages 142-1438, 1996), eg, oral, rectal Administration by subcutaneous implantation or administration by implantation at a desired target site is possible. Sustained release formulations can include polypeptides, polynucleotides, antibodies contained in a reservoir surrounded by a polypeptide, polynucleotide, antibody, and / or rate controlling membrane dispersed in a base matrix. The base used in such formulations is biocompatible and may be biodegradable. This formulation preferably has a relatively constant level of active ingredient release. Bases include microparticles such as poly (lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other delayed release bases include supramolecular bios that include a non-liquid hydrophilic core (eg, a cross-linked polysaccharide or oligosaccharide) and optionally further include an outer layer containing an amphiphilic compound (eg, phospholipid). Vectors (see for example US Pat. No. 5,151,254, PCT applications WO 94/20078, WO 94/23701, WO 96/06638). The amount of active compound contained in a sustained release formulation will vary depending on the site of implantation, the rate of release, the expected duration of release, and the nature of the condition being treated or prevented.

  Any of a number of known delivery vehicles can be used in pharmaceutical compositions and vaccines to facilitate an antigen-specific immune response that targets cells. There is an antigen presenting cell (APC) as a delivery vehicle. Specifically, they are dendritic cells, macrophages, B cells, monocytes, and other cells that may be effective APCs. Such cells may modify genes to increase antigen presentation capacity and / or improve activation and / or maintenance of T cell responses and / or have anti-targeting effects themselves, and / or It can be made immunocompatible with the recipient (ie the person with the matched HLA haplotype), but it is not necessary to do so. APC can generally be isolated from any of a variety of body fluids and organs (including tumor tissue, peritumor tissue). APC can be any of autologous cells, allogeneic cells, syngeneic cells, and heterologous cells.

  In some preferred embodiments of the invention, dendritic cells or progenitor cells thereof are used as antigen presenting cells. Dendritic cells are very powerful APCs (Banchereau and Steinman, Nature, 392, 245-251, 1998) and are effective as physiological adjuvants to elicit anti-tumor immunity for prevention or treatment purposes (See Timmerman and Levy, Ann. Rev. Med., 50, 507-529, 1999). In general, dendritic cells have a typical shape (star-shaped in situ, with a well-characterized cytoplasmic process (dendrites) in vitro) and the ability to capture, process, and present antigens with high efficiency. And the ability to activate naive T cell responses. Dendritic cells, of course, can be genetically engineered to express special cell surface receptors or ligands not commonly found in dendritic cells in vivo or in vitro. The present invention contemplates dendritic cells so modified. Instead of dendritic cells, dendritic cells with secreted vesicle antigens (called exosomes) can also be used in vaccines (Zitvogel et al., Nature Med., 4, 594-600, 1998). See year).

  Dendritic cells and their progenitor cells can be obtained from peripheral blood, bone marrow, tumor infiltrating cells, peritumoral tissue infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood and any other suitable tissue or body fluid . For example, dendritic cells can be differentiated in vitro by adding a combination of cytokines (eg GM-CSF, IL-4, IL-13, TNF-α) to monocyte cultures collected from peripheral blood Can do. Alternatively, CD34 positive cells recovered from peripheral blood, umbilical cord blood, bone marrow can be differentiated, matured from GM-CSF, IL-3, TNF-α, CD40 ligand, LPS, flt3 ligand, and / or dendritic cells They can be differentiated into dendritic cells by adding to the medium a combination of other compounds that induce proliferation.

  Dendritic cells are classified for convenience as "immature" cells and "mature" cells. This is a simple way to distinguish between two well-characterized phenotypes. However, this naming should not be considered to cover all possible intermediate stages of differentiation. Immature dendritic cells are characterized by being APC with a high capacity for antigen uptake and processing. This is associated with high expression of Fcγ receptor and mannose receptor. The mature phenotype is generally less in terms of the expression of these markers, but is important for T cell activation (class I and class II MHC, adhesion molecules (eg CD54, CD11), costimulatory molecules ( For example, CD40, CD80, CD86, 4-1BB)) are often expressed.

  In general, APCs are transfected with a polynucleotide encoding an antigenic polypeptide (or part or variant thereof) so that the antigenic polypeptide or immune part thereof is expressed on the cell surface. Such transfection can be performed in vitro. Compositions or vaccines comprising the cells thus transfected and the adjuvants described herein can be used for therapeutic purposes. Alternatively, a gene delivery vehicle that targets dendritic cells or other antigen-presenting cells can be administered to the patient so that transfection occurs in vivo. For example, transfection of dendritic cells in vivo and in vitro is generally performed by any method known in the art (eg, the method described in WO 97/24447), or Mahvi et al. (Immunology and Cell Biology, 75, pages 456-460, 1997). To make dendritic cells with antigen, the dendritic cells or their progenitor cells are incubated with either the antigen polypeptide, DNA (naked or contained in a plasmid vector), or RNA. Alternatively, it may be incubated with a recombinant bacterium or recombinant virus expressing the antigen (eg, vaccinia vector, fowlpox vector, adenovirus vector, lentivirus vector). Before the dendritic cell is accompanied by the antigen, the polypeptide may be covalently bound to an immune partner serving as a T cell supporter (for example, a carrier molecule). Alternatively, non-binding immune partners can be pulsed to dendritic cells separately from the polypeptide or in the presence of the polypeptide.

Treatment of Nitric Oxide-Related Diseases One aspect of the present invention provides a method of treating a disease or condition caused by nitric oxide, particularly ischemia or reperfusion injury. This method involves administering an effective amount of a compound of the present invention to a patient in need of such treatment. It is generally accepted that inducers of transcription and protein synthesis for the iNOS gene are somewhat toxic to promote inflammation and have little tolerance to animals and humans. Endotoxins (LPS) and inflammatory cytokines (eg, IL-1, TNF-α, IFN-γ) are known to be iNOS inducers. All of which are inherently toxic and can induce a systemic inflammatory response, adult respiratory enhancement syndrome, multiple organ failure, and cardiovascular collapse when administered to animals.

  By examining the cardioprotective activity of MPL® immunostimulatory agents, it was found that induction of nitric oxide synthase (iNOS) is important in the delayed cardioprotective effect of the compounds of the present invention. Furthermore, nitric oxide (NO) signal transduction, possibly occurring through a constitutive pool of NOS, is important in the acute cardioprotective effects by the compounds of the present invention. In view of the residual endotoxin-like activity of MPL® immunostimulants, it is not surprising that the compounds of the present invention may be able to induce nitric oxide signaling. Furthermore, nitric oxide signaling has been suggested to be a potential pathway for pre-control of ischemia and elicit cardioprotection. This finding combined with the fact that nitric oxide donors have a cardioprotective effect further supports that the NOS / NO pathway is the pathway that the MPL® immunostimulant protects the heart. .

  The compounds of the present invention are useful in methods of treating diseases or conditions that are altered or ameliorated by nitric oxide, especially ischemia and reperfusion injury (cardioprotective effects of aminoalkylglucosaminidolinic acid and assays of cardioprotective properties). (See US Patent Application Serial No. 09/808669, filed March 14, 2001, for the law).

The following examples are illustrative and the scope of the present invention is not limited to these examples.
Example 1
N-[(R) -3-tetradecanoyloxytetradecanoyl]-(S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2-[(R) -3-tetradecanoyl Oxytetradecanoylamino] -3-O-[(R) -3-tetradecanoyloxytetradecanoyl] -β-D-glucopyranoside triethylammonium salt; triethylammonium salt of the compound represented by the general formula (II) Preparation

(1a) To dry 1,2-dichloroethane (10 ml), 2-deoxy-4-O-diphenylphosphono-3-O-[(R) -3-tetradecanoyloxytetradecanoyl] -6-O- ( Dissolve 2,2,2-trichloro-1,1-dimethylethoxycarbonyl) -2- (2,2,2-trichloroethoxycarbonylamino) -β-D-glucopyranosyl bromide (1.05 g, 0.81 mmol) 4 angstrom molecular sieve (0.5 g), anhydrous CaSO ₄ (2.2 g, 16 milliol), N-[(R) -3-tetradecanoyloxytetradecanoyl]-(S) -2-pyrrolidinemethanol (0.40 g, 0.75 mmol) was added. The resulting mixture was stirred at room temperature for 1 hour, treated with Hg (CN) ₂ (1.02 g, 4.05 mmol), heated to reflux in the dark for 16 hours. The reaction mixture was cooled, diluted with CH ₂ Cl ₂ and filtered. The filtrate was washed with 1N aqueous KI, dried (Na ₂ SO ₄ ) and concentrated. N-[(R) -3-tetradecanoyloxytetradecanoyl]-(S) -2-pyrrolidinylmethyl by flash chromatography on silica gel (gradient eluent, 15 → 20% EtOAc / hexane) 2-Deoxy-4-O-diphenylphosphono-3-O-[(R) -3-tetradecanoyloxytetradecanoyl] -6-O- (2,2,2-trichloro-1,1-dimethyl 0.605 g (43%) of ethoxycarbonyl) -2- (2,2,2-trichloroethoxycarbonylamino) -β-D-glucopyranoside was obtained as an amorphous solid.

(1b) A solution of the compound prepared in (1a) (0.50 g, 0.29 mmol) dissolved in AcOH (10 ml) at 60 ° C. was divided into three equal parts of zinc dust (0.98 g, 15 mmol) over 1 hour. Processed. The reaction mixture was cooled, sonicated, filtered through a celite pad, and concentrated. The resulting residue was partitioned between CH ₂ Cl ₂ and saturated aqueous NaHCO ₃ and separated into layers. The organic layer was dried (Na ₂ SO ₄ ) and concentrated. A solution of the obtained crude amino alcohol and (R) -3-tetradecanoyloxytetradecanoic acid (0.155 g, 0.34 mmol) in CH ₂ Cl ₂ (3.5 ml) was converted into a powdered 4 Å molecular sieve (0.25 After stirring with g) for 0.5 h, it was treated with 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (0.11 g, 0.44 mmol). The resulting mixture was stirred at room temperature for 8 hours, filtered through celite and concentrated. Flash chromatography on silica gel using 50% EtOAc / hexanes revealed that N-[(R) -3-tetradecanoyloxytetradecanoyl]-(S) -2-pyrrolidinylmethyl 2-deoxy- 4-O-Diphenylphosphono-2-[(R) -3-tetradecanoyloxytetradecanoylamino] -3-O-[(R) -3-tetradecanoyloxytetradecanoyl] -β-D -0.355 g (68%) of glucopyranoside was obtained as a colorless syrup.

(1c) A solution of the compound prepared in (1b) above (0.300 g, 0.166 mmol) in a mixture of AcOH (1 ml) and tetrahydrofuran (9 ml) was dissolved at room temperature in the presence of PtO ₂ (0.15 g). Hydrogenated at 70 psig for 18 hours. The reaction mixture was diluted with CHCl ₃ -MeOH (2: 1, 50 ml) and sonicated briefly. The catalyst was collected and washed with CHCl ₃ -MeOH (2: 1), and the combined filtrate and washings were concentrated. Flash chromatography on silica gel with CHCl ₃ -MeOH-H ₂ O-Et ₃ N (90: 10: 0.5: 0.5) gave a partially purified product that was The solution was dissolved in CHCl ₃ -MeOH (2: 1, 30 ml) cooled in 1 and washed with 0.1N aqueous HCl (12 ml) cooled with ice. The organic phase was filtered and lyophilized from 2% Et ₃ N aqueous solution (5 ml, no pyrogen) to give N-[(R) -3-tetradecanoyloxytetradecanoyl]-(S) -2-pyrroline. Dinylmethyl 2-deoxy-4-O-phosphono-2-[(R) -3-tetradecanoyloxytetradecanoylamino] -3-O-[(R) -3-tetradecanoyloxytetradecanoyl ] -β-D-glucopyranoside triethylammonium salt was obtained as a colorless powder, 0.228 g (79%): melting point 67-70 ° C .; IR (film) 3306, 2955, 2923, 2853, 1736, 1732, 1644, 1548 , 1466, 1378, 1245, 1177, 1110, 1053, 844 cm ^-1 ; ¹ H NMR (CDCl ₃ -CD ₃ OD) δ 0.88 (m, 18H), 1.0−2.05 (mH), 2.20−2.70 (m , 12H), 3.06 (q, 6H, J = 7.2Hz), 3.3−3.25 (mH), 4.52 (d, 1H, J = 8Hz), 5.05−5.28 (m, 4H), 7.44 (d, 1H, J ^{= 9Hz); 13 C NMR (} CDCl 3) δ173.3,173.0,170.3,169.6,168.6,101.8,100.4,75.8,72.5,72.4,70.9,70.8,70.3,70.2,69.9,69.3,67.9 66.6, 56.5, 56.3, 54.5, 47.4, 45.8, 44.6, 41.4, 41.0, 39.7, 39.2, 39.0, 34.5, 34.3, 34.1, 32.0, 29.7, 29.4, 28.1, 27.3, 25.7, 25.3, 25.2, 25.1, 24.0, 22.7, 21.6, 14.1, 8.6.

Calculated for C ₁₀₁ H ₁₉₄ N ₃ O ₁₇ P · H ₂ O: C, 68.47; H, 11.15; N, 2.37; P, 1.75. Actual analysis: C, 68.79; H, 11.00; N, 2.24; P, 1.97.

Example 2
N-[(R) -3-Dodecanoyloxytetradecanoyl]-(S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2-[(R) -3-dodecanoyloxytetra Decanoylamino] -3-O-[(R) -3-dodecanoyloxytetradecanoyl] -β-D-glucopyranoside triethylammonium salt; Preparation of triethylammonium salt of the compound represented by the general formula (III)

(2a) 2-Deoxy-4-O-diphenylphosphono-3-O-[(R) -3-dodecanoyloxytetradecanoyl] -6-O- () in dry 1,2-dichloroethane (3.2 ml) Dissolve 2,2,2-trichloro-1,1-dimethylethoxycarbonyl) -2- (2,2,2-trichloroethoxycarbonylamino) -α-D-glucopyranosyl bromide (1.60 g, 1.27 mmol) 4 angstrom molecular sieve (0.6 g), anhydrous CaSO ₄ (1.0 g, 7.3 mmol), N-[(R) -3-dodecanoyloxytetradecanoyl]-(S) -2-pyrrolidinemethanol ( 0.58 g, 1.14 mmol) was added. The resulting mixture was stirred at room temperature for 1 hour, treated with Hg (CN) ₂ (0.58 g, 2.3 mmol), heated to reflux in the dark for 6 hours. The reaction mixture was cooled, diluted with CH ₂ Cl ₂ and filtered through a celite bed. The filtrate was washed with 1N aqueous KI, dried (Na ₂ SO ₄ ) and concentrated. Flash chromatography on silica gel (gradient eluent, 25 → 35% EtOAc / hexanes) gave N-[(R) -3-dodecanoyloxytetradecanoyl]-(S) -2-pyrrolidinylmethyl 2 -Deoxy-4-O-diphenylphosphono-3-O-[(R) -3-dodecanoyloxytetradecanoyl] -6-O- (2,2,2-trichloro-1,1-dimethylethoxycarbonyl ) -2- (2,2,2-trichloroethoxycarbonylamino) -β-D-glucopyranoside was obtained as a colorless oil, 1.72 g (82%).

(2b) A solution of the compound prepared in (2a) above (1.58 g, 0.806 mmol) dissolved in AcOH (40 ml) at 60 ° C. was divided into three equal parts of zinc dust (2.6 g, 40 mmol) over 1 hour. Processed. The reaction mixture was cooled, sonicated, filtered through a celite pad, and concentrated. The resulting residue was partitioned between EtOAc and saturated aqueous NaHCO ₃ and the layers separated. The organic layer was washed with brine, dried (Na ₂ SO ₄ ) and concentrated to give 1.3 g of a white solid. A solution of the obtained crude amino alcohol and (R) -3-dodecanoyloxytetradecanoic acid (0.45 g, 1.05 mmol) in CH ₂ Cl ₂ was dissolved in 2-ethoxy-1-ethoxycarbonyl-1,2-dihydro Treated with quinoline (0.30 g, 1.21 mmol). The resulting mixture was stirred at room temperature for 18 hours and concentrated. Flash chromatography on silica gel using 40 → 50% EtOAc / hexanes revealed that N-[(R) -3-dodecanoyloxytetradecanoyl]-(S) -2-pyrrolidinylmethyl 2-deoxy -4-O-diphenylphosphono-2-[(R) -3-dodecanoyloxytetradecanoylamino] -3-O-[(R) -3-dodecanoyloxytetradecanoyl] -β-D- 0.89 g (56%) of glucopyranoside was obtained as a white foam.

(2c) A solution of the compound prepared in (2b) above (0.75 g, 0.44 mmol) in a mixture of AcOH (4.5 ml) and tetrahydrofuran (45 ml) was added to room temperature in the presence of PtO ₂ (0.45 g). And hydrogenated at 70 psig for 18 hours. The reaction mixture was diluted with CHCl ₃ -MeOH (2: 1, 50 ml) and sonicated briefly. The catalyst was collected and washed with CHCl ₃ -MeOH (2: 1), and the combined filtrate and washings were concentrated. Partial purification by flash chromatography on silica gel using CHCl ₃ -MeOH-H ₂ O-Et ₃ N (gradient eluent; 96: 4: 0.3: 0.3 → 90: 10: 0.5: 0.5) The product obtained was obtained (0.51 g), which was dissolved in ice-cooled CHCl ₃ -MeOH (2: 1, 50 ml) and washed with ice-cold 0.1N aqueous HCl (20 ml). The organic phase was filtered and concentrated. The resulting white wax was lyophilized from 2% Et ₃ N aqueous solution (70 ml, no pyrogen) to give N-[(R) -3-dodecanoyloxytetradecanoyl]-(S) -2-pyrrole Dinylmethyl 2-deoxy-4-O-phosphono-2-[(R) -3-dodecanoyloxytetradecanoylamino] -3-O-[(R) -3-dodecanoyloxytetradecanoyl]- 0.54 g (78%) of β-D-glucopyranoside triethylammonium salt was obtained as a white powder: mp 146-151 ° C .; IR (film) 3292, 3100, 2958, 2922, 2852, 1739, 1731, 1659, 1651 , 1644, 1562, 1555, 1468, 1455, 1433, 1377, 1339, 1310, 1253, 1238, 1183, 1160, 1107, 1080, 1047, 960, 856, 722 cm ^-1 ; ¹ H NMR (CDCl ₃ -CD ₃ OD) δ 0.88 (m, 18H), 1.0−2.10 (mH), 2.20−2.75 (m, 12H), 3.04 (q, 6H, J = 7.2Hz), 3.3−4.3 (mH), 4.45 (d , 1H, J = 8.5Hz), 5.0-5.28 (m, 4H); 13 C NMR (CDCl 3) δ173.9,173.4,173.2,170.6,170.1,169.2,101.4,75.5,74.0 70.8, 70.7, 70.2, 68.5, 60.5, 56.6, 53.6, 47.4, 45.6, 40.9, 39.6, 38.8, 34.5, 34.3, 34.2, 34.1, 31.9, 29.7, 29.6, 29.5, 29.4, 29.4, 29.3, 29.2, 27.3, 25.2, 25.0, 23.6, 22.7, 21.6, 14.0, 8.3.

The calculated MALDI-MS value for [M + Na] ⁺ was 1590.1900, and the measured value was 1590.1866. Calculated for C ₉₅ H ₁₈₂ N ₃ O ₁₇ P · 3H ₂ O: C, 66.20; H, 10.99; N, 2.44. Actual analysis: C, 66.36; H, 10.69; N, 2.15.

Example 3
N-[(R) -3-decanoyloxytetradecanoyl]-(S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2-[(R) -3-decanoyloxytetra Decanoylamino] -3-O-[(R) -3-decanoyloxytetradecanoyl] -β-D-glucopyranoside triethylammonium salt; Preparation of triethylammonium salt of the compound represented by the general formula (IV)

(3a) 2-Deoxy-4-O-diphenylphosphono-3-O-[(R) -3-decanoyloxytetradecanoyl] -6-O- (in dry 1,2-dichloroethane (3.5 ml) Dissolve 2,2,2-trichloro-1,1-dimethylethoxycarbonyl) -2- (2,2,2-trichloroethoxycarbonylamino) -α-D-glucopyranosyl bromide (1.70 g, 1.38 mmol) 4 angstrom molecular sieve (0.6 g), anhydrous CaSO ₄ (1.2 g, 8.8 mmol), N-[(R) -3-decanoyloxytetradecanoyl]-(S) -2-pyrrolidinemethanol ( 0.60 g, 1.24 mmol) was added. The resulting mixture was stirred at room temperature for 1 hour, treated with Hg (CN) ₂ (0.63 g, 2.5 mmol), heated to reflux in the dark for 6 hours. The reaction mixture was cooled, diluted with CH ₂ Cl ₂ and filtered through a celite bed. The filtrate was washed with 1N aqueous KI, dried (Na ₂ SO ₄ ) and concentrated. Flash chromatography on silica gel (gradient eluent, 25 → 40% EtOAc / hexanes) gave N-[(R) -3-decanoyloxytetradecanoyl]-(S) -2-pyrrolidinylmethyl 2 -Deoxy-4-O-diphenylphosphono-3-O-[(R) -3-decanoyloxytetradecanoyl] -6-O- (2,2,2-trichloro-1,1-dimethylethoxycarbonyl ) -2- (2,2,2-trichloroethoxycarbonylamino) -β-D-glucopyranoside was obtained as a colorless oil, 1.82 g (80%).

(3b) A solution of the compound prepared in (3a) above (1.67 g, 1.02 mmol) in AcOH (50 ml) at 60 ° C. was divided into three equal parts of zinc dust (3.33 g, 51 mmol) over 1 hour. Processed. The reaction mixture was cooled, sonicated, filtered through a celite pad, and concentrated. The resulting residue was partitioned between EtOAc and saturated aqueous NaHCO ₃ and the layers separated. The organic layer was washed with brine, dried (Na ₂ SO ₄ ) and concentrated to give 1.25 g of a white solid. A solution of the obtained crude amino alcohol and (R) -3-decanoyloxytetradecanoic acid (0.53 g, 1.33 mmol) in CH ₂ Cl ₂ was dissolved in 2-ethoxy-1-ethoxycarbonyl-1,2-dihydro Treated with quinoline (0.38 g, 1.53 mmol). The resulting mixture was stirred at room temperature for 18 hours and concentrated. Flash chromatography on silica gel using 40 → 50% EtOAc / hexanes revealed that N-[(R) -3-decanoyloxytetradecanoyl]-(S) -2-pyrrolidinylmethyl 2-deoxy -4-O-diphenylphosphono-2-[(R) -3-decanoyloxytetradecanoylamino] -3-O-[(R) -3-decanoyloxytetradecanoyl] -β-D- 1.23 g (74%) of glucopyranoside was obtained as a white foam.

(3c) A solution of the compound prepared in (3b) above (1.07 g, 0.654 mmol) in a mixture of AcOH (6.5 ml) and tetrahydrofuran (65 ml) was added to room temperature in the presence of PtO ₂ (0.66 g). And hydrogenated at 70 psig for 18 hours. The reaction mixture was diluted with CHCl ₃ -MeOH (2: 1, 50 ml) and sonicated briefly. The catalyst was collected and washed with CHCl ₃ -MeOH (2: 1), and the combined filtrate and washings were concentrated. The obtained waxy solid was lyophilized from a 2% aqueous triethylamine solution to obtain about 1 g of a crude triethylammonium salt as a white powder. Partial purification by flash chromatography on silica gel with CHCl ₃ -MeOH-H ₂ O-Et ₃ N (gradient eluent; 96: 4: 0.3: 0.3 → 88: 12: 1: 0.6) The product obtained was obtained (0.84 g), which was dissolved in ice-cooled CHCl ₃ -MeOH (2: 1, 168 ml) and washed with ice-cold 0.1 N aqueous HCl (67 ml). The organic phase was filtered and concentrated. The resulting white wax (approximately 0.7 g) was lyophilized from 2% Et ₃ N aqueous solution (70 ml, no pyrogen) to give N-[(R) -3-decanoyloxytetradecanoyl]-(S ) -2-Pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2-[(R) -3-decanoyloxytetradecanoylamino] -3-O-[(R) -3-decanoyloxy Tetradecanoyl] -β-D-glucopyranoside triethylammonium salt was obtained as a white powder, 0.79 g (79%): mp 121-122 ° C .; IR (film) 3287, 3093, 2961, 2913, 2850, 1745, 1738, 1732, 1716, 1666, 1660, 1651, 1644, 1635, 1565, 1556, 1538, 1470, 1455, 1434, 1416, 1378, 1337, 1311, 1248, 1184, 1104, 1081, 1021, 964, 721 cm ^-1 ; ¹ H NMR (CDCl ₃ -CD ₃ OD) δ 0.88 (m, 18H), 1.0−2.05 (mH), 2.20−2.75 (m, 12H), 3.04 (q, 6H, J = 7.2 Hz) 3.3-4.3 (mH), 4.45 (d, 1H, J = 8.5 Hz), 5.0-5.28 (m, 4H); ¹³ C NMR (CDCl ₃ ) δ 173.7, 173.4, 173.2, 170.5, 170.1, 16 9.1, 101.4, 75.6, 74.0, 70.8, 70.2, 68.7, 60.4, 56.6, 53.8, 47.4, 45.6, 41.0, 39.6, 38.9, 34.5, 34.3, 34.2, 34.1, 31.9, 29.7, 29.6, 29.5, 29.4, 29.4, 29.3, 29.2, 27.3, 25.3, 25.0, 23.7, 22.7, 21.6, 14.1, 8.4.

The calculated MALDI-MS value for [M + Na] ⁺ was 1506.0961, and the measured value was 1506.1008. C ₈₉ H ₁₇₀ N ₃ O ₁₇ Calculated for P: C, 67.43; H, 10.81; N, 2.65. Actual analysis: C, 67.26; H, 10.85; N, 2.47.

Examples 4-8
The main purpose of Examples 4-8 was to generate fever when the compound of general formula (II) (hereinafter referred to as “Compound II”) prepared in Example 1 (as triethylamine salt) was formulated with the vaccine antigen. It was to clarify whether the activity of the adjuvant can be transmitted with as little as possible.

Example 4
Activity of adjuvants against HBsAg (hepatitis B surface antigen) Multiple groups of BALB / c mice (Jackson Laboratories, Bar Harbor, Maine), 6-8 weeks of age, were treated with 2 μg of HBsAg (Laboratorio Pablo Cassala) ± 20 μg of adjuvant (MPL® immunostimulator or compound II) was injected subcutaneously on day 0 and day 21. A vaccine was prepared by mixing recombinant HBsAg with a TEoA (triethanolamine) formulation containing an adjuvant. Sera collected and pooled 21 days after the second vaccination (5 mice per group) revealed titers against HBsAg by ELISA (Table 1). The control non-immunized group was not vaccinated.

  Serum from mice that received Compound II had a significantly greater anti-HBsAg response than control serum that received antigen alone (Table 1). Of particular note was the increase in titer for the IgG2a and IgG2b isotypes. These titers were equivalent to those developed in the control group that received the MPL® immunostimulator.

Example 5
Adjuvant activity against hemagglutinin protein in FluZone influenza vaccine Multiple groups of BALB / c mice (Jackson Laboratories, Bar Harbor, Maine), 6-8 weeks of age, were treated with FluZone influenza vaccine (Connaught • 0.2 μg hemagglutinin protein ± 20 μg adjuvant (MPL® immunostimulatory or compound II) in Laboratories, Swiftwater, PA) was injected subcutaneously on days 0 and 14 . FluZone ELISA revealed FluZone titers from pooled sera collected from 5 mice 14 days after the second vaccination (Table 2). The control non-immunized group was not vaccinated. The first dilution used for sera from the test group was 1: 1600.

  The result was similar to the above example. Again, the potency of Compound II was significantly greater than that of the control sera to which only the antigen was administered (Table 2). The increase in titer was also reflected in the increased response of IgG2a and IgG2b. These titers were equivalent to those developed in the control group that received the MPL® immunostimulator.

Example 6
Activity of adjuvants against HBsAg Multiple groups of BALB / c mice received 2.0 μg HBsAg (Line Americana and Line Biotech) ± 25 μg adjuvant (MPL® immune promoter or compound II) on day 0 And subcutaneous injection on day 21. Sera collected and pooled on day 21 after the second vaccination revealed the IgG1 and IgG2a isotype titers against HBsAg by ELISA (Table 3). The control non-immunized group was not vaccinated. In this experiment, in the case of Compound II, the titer increased compared to the control group to which PBS containing the antigen was administered. RC-553 showed a potency comparable to that of the positive control MPL® immunostimulator (Table 3).

Example 7
Compound II increases CTL activity against mice immunized with HBsAg Several mice belonging to each group of Example 4 were also used as spleen cell donors to evaluate CTL activity. HBsAg directed specific lysis was assessed in a standard 4 hour ⁵¹ Cr release assay (Moore et al., 1988, Cell 55, 777-785). Single cell suspensions were prepared from the spleens of mice 9 days after vaccination. Treat spleen cells with Tris-buffered NH ₄ Cl solution to remove erythrocytes, then in RPMI / 10% FCS supplemented with 5 mM HEPES, 4 mM L-glutamine, 0.05 mM 2-mercaptoethanol, antibiotics. It was dispersed again at a concentration of 7.5 × 10 ⁶ cells / ml. A synthetic peptide known as a representative MHC class I L ^d- restricted CTL epitope (IPQSLDSWWTSL) was added to these cells to a final concentration of 75 nM. After incubation for 4 days, cells were harvested and CTL activity was assessed. Specific killing power against transfected ⁵¹ Cr-labeled P815S cells expressing the above L ^d restricted CTL epitopes was measured. Target cells express L ^d restricted CTL epitopes were transfected P815 cell line (P815S). Nonspecific lysis to the P815 target was less than 10% when the effector: target ratio was 50: 1 (Table 4). Unlike the antibody response, RC553 significantly increased the level of CTL activity compared to the antigen-only control (Table 4).

Example 8
In vitro cytokine induction by Compound II The effect of Compound II on the production of TNF-α and IL-1β was measured in mononuclear cells of human peripheral blood in vitro. MPL (registered trademark) immunostimulant and Compound II were dissolved in 0.2% TEoA / WFI aqueous solution to prepare a preparation.

  Human whole blood was used to evaluate the ability of glycolipid (AGP) to induce inflammatory cytokines. Human whole blood was collected in heparinized tubes and 0.45 ml whole blood was mixed with 0.05 ml phosphate buffer solution (PBS, pH 7.4) containing glycolipid (ie test compound). The tubes were incubated for 4 hours at 37 ° C. on a shaker. Samples were then diluted with 1.5 ml of sterile PBS and centrifuged. By removing the supernatant and performing sandwich ELISA using R & D Systems' Quantickin immunoassay kit to search for human TNF-α and IL-1β, cell-related TNF-α and IL-1β Analyzed whether it exists.

  In the assay, at 1 μg / ml, 5 μg / ml, and 10 μg / ml, Compound II did not produce detectable levels of TNF-α under the assay conditions. Conversely, the positive control MPL® immunostimulant was able to induce TNF-α in the concentration range of 100-10,000 ng / ml.

  Similarly, Compound II (1 μg / ml, 5 μg / ml, 10 μg / ml) did not produce detectable levels of IL-1β. As a standard for comparing the effects of this compound, assuming that the level of IL-1β induced using an MPL (registered trademark) immunostimulator is 1, the relative induction of cytokine by Compound II was 0.05 or less.

Discussion of Examples 4-8 The data from these studies indicate that Compound II can increase immunity against vaccine antigens. This compound increased serum titers against two different vaccine antigens: influenza antigen and hepatitis surface antigen. Like the MPL® immunostimulant, this compound shifted the antibody profile from a response with a predominance of IgG1 isotype to a response with high levels of IgG2a antibody. In addition to increasing the antibody response, this compound is also a good adjuvant for inducing CTL activity.

  Of note in the results of this study is that compound II appears to affect the response without inducing the proinflammatory cytokines TNF-α and IL-1β to verifiable levels. is there. Both of these cytokines are produced by cells of the innate immune system in response to bacterial cell wall products (including lipid A). Since compound II is similar in structure to lipid A, it is also thought to stimulate TNF-α or IL-1β, and indeed many AGP molecules stimulate TNF-α or IL-1β. TNF-α and IL-1β, as inflammatory cytokines, stimulate other cytokine mediators that are important for phagocytic cell activation and activation of specific immunity and trigger the cascade. IL1 was originally called an endogenous pyrogen because it induces a fever response. Thus, if no detectable IL-1 is found after administration of Compound II, the rabbit pyrogen test does not cause fever.

  In these studies, this compound actually promotes the secretion of TNF-α and IL-1β at levels sufficient to activate specific immunity. It can be considered too low to detect in the assay. Another way of thinking would be that this compound stimulates cytokine mediators other than TNF-α and IL-1β, resulting in a specific immune response against simultaneously administered vaccine antibodies. It appears that IFN-γ is being produced. This cytokine is thought to be involved in inducing isotype switching and switching to an IgG2a subclass antibody and a Th-1 type CTL response promoter. Thus, the increase in IgG2a titer and active CTL assembly both reflect the production of IFN-γ.

Example 9
Stimulation of Carbon Monoxide Synthase (iNOS) Inducible by Compound II This example demonstrates the effect of various glycolipids on iNOS induction in J774 murine macrophages. The murine macrophage cell line J774 can be sensitized with IFN-γ in vitro and responds very well to subsequent upregulation of iNOS by stimulation with LPS. This response is measured by a standard Glyce reagent ELISA assay. In this assay, J774 cells are placed in a 30 ml flask at a rate of 1 × 10 ⁶ cells / ml and IFN-γ is added at a rate of 100 units / ml over 16-24 hours. Cells are then harvested, washed, redispersed at a rate of 2 × 10 ⁵ cells / well in a 96-well plate, and allowed to adhere. Glycolipid compounds are serially diluted and placed into wells for the test group, and the resulting culture is incubated for an additional 36-40 hours, after which the culture supernatant is collected and the Gryce reagent for nitrite release Analysis is performed (Green et al., 1982, Anal. Biochem., 126, 131-138). Nitrite content is closely related to iNOS function.

Potency was determined as the concentration of glycolipid (ng / ml) that could induce 50% of the maximum value of nitrite induced in the culture (ED ₅₀ ). The smaller the ED ₅₀ value, the greater the potency of inducing iNOS. The ED ₅₀ was calculated according to the method described in Johnson et al., J. Med. Chem., 42, 4640-4649, 1999.

The MPL® immunostimulant was found to have an ED ₅₀ of about 2 ng / ml. This produces a high level of nitrite. In contrast, Compound II had an ED ₅₀ of about 3000 (ng / ml) or more.

  The observation that the maximum activity of iNOS is very low with this compound suggests that this compound is essentially inactive with respect to induction of iNOS in this system.

  The examples and embodiments described in this specification are for illustrative purposes, and various modifications and changes can be proposed to those skilled in the art based on the examples and embodiments. Accordingly, such modifications and changes are considered to be within the spirit and scope of this specification and the scope of the appended claims. All publications, patents, and patent applications cited in this specification are actually incorporated herein in their entirety for reference.

Claims (24)

General formula:

And a pharmacologically acceptable salt thereof, provided that
X is selected from the group consisting of -O- and -NH-;
Y is selected from the group consisting of -O- and -S-;
R ¹ , R ² , R ³ are each independently selected from the group consisting of (C ₉ -C ₁₃ ) acyl;
R ⁴ is —H and —PO ₃ R ⁷ R ⁸ (where R ⁷ and R ⁸ are each independently selected from the group consisting of —H and (C ₁ -C ₄ ) aliphatic groups) Select from the group
R ⁵ is —H, —CH ₃ , —PO ₃ R ⁹ R ¹⁰ (where R ⁹ and R ¹⁰ are independently from the group consisting of —H and (C ₁ -C ₄ ) aliphatic groups. Select from the group consisting of;
R ⁶ is H, OH, (C ₁ -C ₄ ) oxyaliphatic group, -PO ₃ R ¹¹ R ¹² , -OPO ₃ R ¹¹ R ¹² , -SO ₃ R ¹¹ , -OSO ₃ R ¹¹ , -NR ¹¹ R ¹² , -SR ¹¹ , -CN, -NO ₂ , -CHO, -CO ₂ R ¹¹ , -CONR ¹¹ R ¹² (where R ¹¹ and R ¹² are independently H and (C _{1 to} C ₄ ) Selected from the group consisting of aliphatic groups), wherein one of R ⁴ and R ⁵ is a phosphorus-containing group and R ⁴ is —PO ₃ R ⁷ R ⁸ In certain instances, R ⁵ is other than —PO ₃ R ⁹ R ¹⁰ ;
“* 1”, “* 2”, “* 3”, “**” represent chiral centers;
n, m, p and q each independently represents an integer of 0 to 6, but the sum of p and m is 0 to 6). X and Y are -O-, R ⁴ is PO ₃ R ⁷ R ⁸ , R ⁵ and R ⁶ are H, and n, m, p, q are integers from 0 to 2. 1. The compound according to 1. The compound according to claim 2, wherein R ⁷ and R ⁸ are -H.   4. The compound according to claim 3, wherein n is 1, m is 2, and p and q are 0. The compound according to claim ¹ , wherein R ¹ , R ² and R ³ are each (C ₁₀ -C ₁₂ ) acyl. 5. The compound according to claim 4, wherein R ¹ , R ² and R ³ are each a decanoyl residue. 5. The compound according to claim 4, wherein R ¹ , R ² and R ³ are each a dodecanoyl residue.   5. The compound according to claim 4, wherein * 1, * 2, and * 3 are R configurations.   A pharmaceutical composition comprising a pharmacologically acceptable base and the compound according to any one of claims 1 to 8.   10. A pharmaceutical composition according to claim 9, comprising a therapeutically effective amount of the compound according to any one of claims 1-8.   The pharmaceutical composition according to claim 9 or 10, further comprising at least one antigen.   The above antigen is herpes simplex virus type 1, herpes simplex virus type 2, human cytomegalovirus, HIV, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, RS (Respiratory Syncytial) Virus, human papilloma virus, influenza virus, Mycobacterium tuberculosis, Leishmania spp., Cruz trypanosoma, Ehrlichia, Candida, Salmonella, Neisseria, Borrelia, Chlamydia, Bordetella, Plasmodium, Toxoplasma 12. The pharmaceutical composition according to claim 11, derived from.   12. The pharmaceutical composition according to claim 11, wherein the antigen is a human tumor antigen.   14. The tumor antigen according to claim 13, wherein the tumor antigen is derived from any of prostate cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, brain tumor, head and neck cancer, melanoma, leukemia, and lymphoma. Pharmaceutical composition.   14. The pharmaceutical composition according to claim 13, wherein the antigen is a self antigen.   16. The pharmaceutical composition according to claim 15, wherein the autoantigen is an antigen related to an autoimmune disease.   17. The pharmaceutical composition according to claim 16, wherein the autoimmune disease is any of type 1 diabetes, multiple sclerosis, myasthenia gravis, rheumatoid arthritis, and psoriasis.   The pharmaceutical composition according to any one of claims 10 to 17, which is in the form of an aqueous preparation.   19. The pharmaceutical composition according to claim 18, wherein the aqueous preparation comprises one or more surfactants.   The pharmaceutical composition according to any one of claims 10 to 17, which is in the form of an emulsion preparation.   The pharmaceutical composition according to any one of claims 10 to 17, which is in the form of a solid preparation.   A method for inducing an immune response in a patient, wherein the patient is treated with a compound according to any one of claims 1-8 or a composition according to any one of claims 9-21. A method comprising an operation of administering an effective amount.   A method for treating a mammal suffering from, or susceptible to, any of pathogenic fungal infections, cancer and autoimmune diseases, wherein the mammal is any one of claims 1 to 8. A method comprising administering a therapeutically effective amount of a compound according to claim 9 or a composition according to any one of claims 9-21.   A method of treating a disease or condition ameliorated by the production of nitric oxide in a patient's body, wherein the patient is treated as a compound according to any one of claims 1-8 or according to claims 9-21. A method comprising contacting with an effective amount of the composition according to any one of the preceding claims.